The effect of TG2-inhibitory monoclonal antibody zampilimab on tissue fibrosis in human in vitro and primate in vivo models of chronic kidney disease

Fibrotic remodeling is the primary driver of functional loss in chronic kidney disease, with no specific anti-fibrotic agent available for clinical use. Transglutaminase 2 (TG2), a wound response enzyme that irreversibly crosslinks extracellular matrix proteins causing dysregulation of extracellular matrix turnover, is a well-characterized anti-fibrotic target in the kidney. We describe the humanization and characterization of two anti-TG2 monoclonal antibodies (zampilimab [hDC1/UCB7858] and BB7) that inhibit crosslinking by TG2 in human in vitro and rabbit/cynomolgus monkey in vivo models of chronic kidney disease. Determination of zampilimab half-maximal inhibitory concentration (IC50) against recombinant human TG2 was undertaken using the KxD assay and determination of dissociation constant (Kd) by surface plasmon resonance. Efficacy in vitro was established using a primary human renal epithelial cell model of tubulointerstitial fibrosis, to assess mature deposited extracellular matrix proteins. Proof of concept in vivo used a cynomolgus monkey unilateral ureteral obstruction model of chronic kidney disease. Zampilimab inhibited TG2 crosslinking transamidation activity with an IC50 of 0.25 nM and Kd of <50 pM. In cell culture, zampilimab inhibited extracellular TG2 activity (IC50 119 nM) and dramatically reduced transforming growth factor-β1-driven accumulation of multiple extracellular matrix proteins including collagens I, III, IV, V, and fibronectin. Intravenous administration of BB7 in rabbits resulted in a 68% reduction in fibrotic index at Day 25 post-unilateral ureteral obstruction. Weekly intravenous administration of zampilimab in cynomolgus monkeys with unilateral ureteral obstruction reduced fibrosis at 4 weeks by >50%, with no safety signals. Our data support the clinical investigation of zampilimab for the treatment of kidney fibrosis.


S1.2
Showing the sequence alignments of the final humanized versions of BB7 and DC1 kappa light chains.Gaps (-) are used to maintain Kabat numbering, and to show residue insertion or deletion where applicable. 2 per timepoint (0 days, n = 2; 7 days, n = 2; 14 days n = 2; 21 days, n = 2; 28 days, n = 2).ECM, extracellular matrix; RPTEC, renal proximal tubule epithelial cells; SD, standard deviation; TG2, transglutaminase 2; TGF-β1, transforming growth factor-β1.Blood (6 mL) was collected for hematology and clinical chemistry before surgery (including sham operations) and again during the last week before termination.One mL blood was collected in EDTA tubes for hematology analysis and the remainder was collected into a plain tube to produce serum for clinical chemistry.

Hematology
The parameters detailed in S2.1 Table (below) were determined using an automatic blood analyzer (SYSMEX XE2100).

Clinical chemistry on serum
The parameters presented in S2.2 Table (below) were determined using automatic blood chemistry analyzer (OLYMPUS AU5400).

Urinalysis
During the early morning, urine samples were collected by putting monkeys in a metabolic cage for 2 hours, once during the pre-test phase and once during the last week of treatment.
Volume and density were recorded; 2 mL were used for semi-quantitative determinations (including protein and glucose as a minimum) by using multistrip, followed by dry chemistry analysis (to compare the color of the different samples]).All determinations presented in S2.3 Methods Table (below) were conducted on fresh samples.

Methods: Pathology Necropsy and tissue preservation
A full macroscopic examination of the collected tissues was performed on animals by the sitting veterinarian under the general supervision of a pathologist, and all lesions were recorded.

Histology/pathology
At study termination, animals were sacrificed by phenobarbital overdose followed by exsanguination.All tissues listed in S2.4 Methods Table (below) were collected, macroscopically examined, and fixed in 10% neutral-buffered formalin.Tissues marked with an X in the column "tissue examined" were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin (H&E).These were subsequently microscopically examined by a study pathologist and any abnormality reported.
Blood smear and bone marrow smear (Groups 1, 2, 4, 5): blood smear and bone marrow smear (sternum) were prepared at termination.Slides were fixed and stained with Wright-Giemsa and examined by the pathologist.A detailed sample collection/preparation procedure is given in the pathology report.

B
Masson's trichrome C Hydroxyproline D TG in situ activity TG2 Activity (Area %) S1.1 Fig. Development of a UUO model in the New Zealand white rabbit.Eight New Zealand white rabbits underwent a left UUO with two animals culled every 7 days thereafter; the kidneys were recovered and analyzed for tubulointerstitial fibrosis and TG activity.Two additional animals received a sham operation.(A) Top row: Masson's trichrome staining showing an exemplar cortical image at x100 magnification from various time point.Blue staining indicates collagen; red/pink indicates cellular areas.Bottom row: corresponding images showing the level of TG activity at each time point.Red staining shows incorporation of TG substrate biotin cadaverine.(B) Quantitative computerized image analysis of whole

(
C) Quantification of total kidney collagen by amino acid analysis of hydroxyproline from acid-hydrolyzed kidney samples at each time point.Each data point signifies a mean value from the analysis of two independent kidney segments from each animal.Data represent mean ± SD hydroxyproline concentration per 20 µL of a 10% renal homogenate.n = 2 per group.(D) Quantification of TG2 activity by cadaverine incorporation on whole kidney scans.Activity was calculated using the proportion of orange versus blue staining across timepoints.Data represent mean TG activity ± SD. n = 2 per timepoint.Scale bar = 75 µm.d, days; OH, hydroxy; SD, standard deviation; TG, transglutaminase; UUO, unilateral ureteral obstruction.% inhibition S1.2 Fig. ECM protein accumulation is inhibited by zampilimab in a primary human renal proximal tubule epithelial cell model of tubulointerstitial fibrosis: percentage inhibition.Percentage inhibition of the accumulation of (A) fibronectin, (B) collagen I and III, (C) collagen IV, and (D) collagen V in the ECM of RPTEC monocultures by zampilimab (red), huBB7 (green) or isotype control (A33 IgG4P; black) in cells induced with TGF-β1 (30 ng/mL).Calculated using the data in Fig 1B.Results are shown from one of four repeat experiments.Data represent mean level of percentage inhibition ± SD from eight technical repeats.ECM, extracellular matrix; Ig, immunoglobulin; RPTEC, renal proximal tubule epithelial cells; TGF-β1, transforming growth factor-β1.
B Regression slope analysis S1.3 Fig. TG2 inhibitory antibody zampilimab inhibits total ECM accumulation in a primary human renal proximal tubule epithelial cell model of tubulointerstitial fibrosis: percentage inhibition.Percentage inhibition of total ECM accumulation (measured using tritiated amino acid incorporation) by zampilimab (0-400 nM) in a primary human RPTEC model of tubulointerstitial fibrosis using induction with TGF-β1 (30 ng/mL).Data (Fig 1C) were calculated as a simple percentage inhibition (A) or log-transformed and a non-linear log (antagonist) versus response variable slope regression analysis (B).Data represent mean percentage inhibition (±SD).ECM, extracellular matrix; RPTEC, renal proximal tubule epithelial cells; SD, standard deviation, TG2, transglutaminase 2; TGF-β1, transforming growth factor-β1. .TG2 inhibitory antibody rbBB7 inhibits total ECM accumulation in a primary rabbit renal proximal tubule epithelial cell and renal fibroblast co-culture model of tubulointerstitial fibrosis: percentage inhibition.Percentage inhibition of total ECM accumulation (as measured by isotopic ECM labelling) by rbBB7 (0.75-200 µg/mL) in a rabbit RPTEC and fibroblast co-culture model of tubulointerstitial fibrosis using induction of ECM by TGF-β1 (10 ng/mL).Data (Fig 2C) were log-transformed and a log (antagonist) versus response variable slope regression analysis performed.Data represent mean percentage inhibition (±SD).
monkey UUO model show expected pharmacokinetics.Measurement of TG2 inhibitory antibodies rBB7 in a rabbit UUO study(Fig 4)  and zampilimab in a cynomolgus monkey UUO(Fig 6)  were made in plasma taken at trough (i.e., immediately before the next injection, which is equivalent to 1 IgG half-life) and in the cynomolgus monkey at additional time points.(A) Rabbit.Trough concentrations of rbBB7 in the UUO + rbBB7 group (n = 4) immediately before dosing at 5-day intervals.Antibody levels were measured by ELISA.The antibody signal at time 0 was below the threshold of A rbBB7 plasma levels in rabbit B Zampilimab plasma levels in cynomolgus monkey Normal + zampilimab 100 mg/kg UUO + zampilimab 50 mg/kg UUO + zampilimab 10 mg/kg detection.Data represent mean plasma concentration of rbBB7 (±SD).(B) Cynomolgus monkey: plasma concentrations of zampilimab were measured by mass spectrometry in the UUO + zampilimab 10 mg/kg (n = 6) and UUO + zampilimab 50 mg/kg (n = 4) groups plus in a normal cynomolgus monkey treated with zampilimab 100 mg/kg (n = 2).Data represent mean plasma concentration of zampilimab (±SD).d, day; ELISA, enzyme-linked immunosorbent assay; Ig, immunoglobulin; SD, standard deviation; TG2, transglutaminase 2; UUO, unilateral ureteral obstruction.A TG2 activity (T26 substrate) S1.6 Fig. rbBB7 is a potent inhibitor of in situ TG2 activity in a rabbit UUO model of CKD.Eight New Zealand white rabbits were subjected to a left UUO and kept for 25 days for tubulointerstitial fibrosis to develop.Four UUO animals received no treatment (UUO), and four received 100 mg/kg of TG2 inhibitory antibody rbBB7 (UUO + rbBB7) every 5 days.Four additional animals received a sham operation.The last dose of rbBB7 was given on Day 20 and measurements of TG activity were taken at termination (5 days, or one IgG half-life later).(A) Representative images showing staining of in situ TG2 activity measured by incorporation of the TG2 preferred substrate T26 peptide (red) in sections from the sham, UUO, and UUO + rbBB7 groups.Cell nuclei are shown in blue.Magnification of total TG activity using the incorporation of the pan TG substrate cadaverine.Data represent mean percentage area of cadaverine incorporation ± SD.Significance was calculated by one-way ANOVA with Tukey's post hoc test.(C) Quantification of TG2 activity based on TG2 preferred substrate T26 peptide incorporation.Data represent mean percentage area of T26 incorporation ± SD.Significance was calculated by t-test.Scale bar = 75 µm.****p<0.00001;ANOVA, analysis of variance; CKD, chronic kidney disease; Ig, immunoglobulin; SD, standard deviation; TG, transglutaminase; UUO, unilateral ureteral obstruction.S1.7 Fig. rbBB7 prevents accumulation of collagen III and fibronectin in a rabbit UUO model of CKD.Eight New Zealand white rabbits were subjected to a left UUO and left for 25 days for tubulointerstitial fibrosis to develop.Four UUO animals received no treatment (UUO) and 4 received 100 mg/kg of TG2 inhibitory antibody rbBB7 (UUO + rbBB7) every 5 days.Four additional animals received a sham operation (sham).Collagen III (A) and fibronectin (B) were measured by immunofluorescence (red) on whole kidney sections, scanned, and the area of positive staining (as a percentage of the area of DAPI stain) measured by computerized high content image analysis using Definiens software.Magnification x200.(A&C) Exemplar images and quantification for collagen III.(B&D) Exemplar images and quantification for fibronectin.Data represent mean percentage area ± SD and significance shown by t-test.Scale bar = 75 µm.01; ***p<0.0005;CKD, chronic kidney disease; DAPI, 4′,6-Diamidino-2-phenylindole dihydrochloride; SD, standard deviation; TG2, transglutaminase 2; UUO, unilateral ureteral obstruction.S1.8 Fig.Effect of zampilimab on dermal wound closure in cynomolgus monkeys.Dermal wounds (6 per animal; 12 treated with zampilimab, 8 untreated) were made in the back skin of cynomolgus monkeys treated once weekly with up to 100 mg/kg IV zampilimab.Wounds were assessed up to 28 days for changes in closure and cell infiltration.Hematoxylin and eosin-stained images were obtained 3 days post injury from one untreated animal (left) and 2 zampilimab-treated animals (50 mg/kg [middle] and 100 mg/kg [right]).Circles indicate where the dermal wound is in the section.The left section (untreated) shows typical closure of the wound, the middle and right sections (zampilimab treated) show partial closure of the wound.Partial closure of a wound at 3 days, as exemplified in the treated animals, was equally prevalent in untreated and zampilimab-treated animals and is within the natural Zampilimab 50 mg/kg Zampilimab 100 mg/kg Untreated variation of early wound closure.Of the wounds examined at 3 days post injury (6 per animal), no clear alteration in wound closure was observed, with typically 2 out of 6 wounds remaining partly open irrespective of treatment.No open wounds were observed after 3 days in any animal.Overall, no clear differences in wound healing between control and zampilimab-treated animals were reported by the pathologist.Scale bar ~1000 μm (top row, low power); ~50 μm (bottom row, high power).IV, intravenous.S1.9 Fig. Zampilimab does not affect normal kidney histology in cynomolgus monkeys.Kidneys from 2 normal cynomolgus monkeys (left) and 2 normal cynomolgus monkeys treated for 28 days with zampilimab 100 mg/kg (right) were sectioned, stained with Masson's Trichrome and examined microscopically.100 x magnification.Scale bar = 75 µm.

Table. Tissue list.
The two animals in the normal + zampilimab 100 mg/kg group showed increased centrilobular glycogen vacuoles (left kidney).In addition, one animal presented bilateral tubular dilation in the renal cortex.The relationship of this change with administration of zampilimab is uncertain given the low number of animals in this group.No macroscopic change was observed at the injection site of any animal; the only microscopic changes observed were minimal and mild hemorrhage and inflammatory cell infiltration, which are commonly observed with the subcutaneous administration of therapeutic antibodies.Due to inadequate tissue sampling in several animals, an accurate evaluation of this tissue was not possible.All other microscopic findings were considered spontaneous changes in Cynomolgus monkeys of this age (see S2.8 Table in the Weight tab of the Supplemental Excel file).out of the normal range during pre-test or at the end of the study but without correlation with UUO surgery or zampilimab treatment (S2.10 Table in the Clinical Chemistry tab of the Supplemental Excel file).Although albumin levels were within the normal range, there was a trend toward decreased levels at study termination in UUO animals and UUO animals treated with zampilimab 10 mg/kg.The decrease was not observed with the 50 mg/kg dose, suggesting a protective effect of the highest dose of zampilimab (S2.11Table in the Clinical Chemistry tab of the Supplemental Excel file).Urea and creatinine levels were increased 4 weeks after UUO surgery in the presence or in the absence of zampilimab (S2.13 Table in the Clinical Chemistry tab of the Supplemental Excel file).By contrast, no significant changes were observed in animals undergoing no ligation.Other parameters were within control ranges and no real effect of UUO or zampilimab treatment could be evidenced.Hematology data indicated no significant effects of UUO surgery and/or zampilimab treatment.The reticulocyte count was elevated in all groups at the end of the study, without clear effect of the UUO surgery and/or the treatment with zampilimab (S2.17 to S2.28 Tables in the Complete Cell Counts tab of the Supplemental Excel file).Urine analyses performed before and after surgery indicated mainly no changes in bilirubin, urobilinogen, ketones, proteins, nitrites determined by dipstick, no changes in pH, color or microscopic examination of sediments.However, blood score (by dipstick) was higher for animals with UUO, with or without zampilimab 10 mg/kg, suggesting kidney damage after UUO and some protection with the 50 mg/kg dose of zampilimab (Supplemental Fig 2.1 below and S2.29 to S2.38 Tables in the Urinalysis tab of the Supplemental Excel file).

Fig. Effect of UUO and weekly zampilimab treatment for 4 weeks on serum levels of urea, creatinine, calcium, and phosphate.
Blood samples (Groups 1, 2, 4 and 5) were collected before (Baseline) and 4 weeks after UUO (at termination), serum were prepared and analyzed.